Technical Abstract:
The diamondback moth (DBM), Plutella xylostella (L.) is one of the most important pests of the cabbage family, as well as other vegetable crops throughout the world. Its control by chemical insecticides as well as the biopesticide Bacillus thuringiensis has become more difficult due to the development of resistance to these agents. The present study was initiated to evaluate the replication of a recombinant baculovirus of DBM (PxMNPV AaIT) developed by American Cyanamid from the parental wild type baculovirus as well as to test its pathogenicity against several lepidopteran species. The recombinant carries the toxin gene engineered into the deleted egt gene site. Cell lines employed in this study were derived from Helicoverpa zea (HZ-FB33), Heliothis virescens (HV-OV), Trichoplusia ni (TN-CL1), Spodoptera frugiperda (SF-TS) and S. exigua (SE-E4). Cells were seeded in triplicate wells in a 24 well plate at 2x105 cells/ml in a volume of 2 ml per well and allowed to attach overnight. Cells were challenged at a multiplicity of infection (MOI) of 1 and placed on a rocker platform for two hours. At the end of this period cells were washed several times with Hanks¿ balanced salts solution (HBSS) and 2 ml of growth medium (ExCellTM containing 10% FBS) added, and inoculated cultures incubated at 280C for 7 days. At the end of this period supernatant fluids from inoculated cultures were recovered by centrifugation and titered employing the tissue culture infectious dose fifty (TCID50) method. Twenty-four old larvae from H. subflexa, H. virescens, H. zea, T.ni and P. xylostella were tested for their susceptibility to the recombinant employing the lethal concentration fifty( LC50) bioassay. Cell cultures were examined several hours after inoculation and on a daily basis for signs of cell toxicity but none were observed. The SE-E4 cell line produced the highest titer of 6.7 + 0.06 x 105 TCID50/ml whereas the greatest number of occlusion bodies (OB) of 1.1 + 12.05 x 107/ml was produced by SF-TS. Of the five lepidopteran species of insects investigated, H. virescens was the most susceptible, with an LC50 of 0.452 OB/cm2, followed by P. xylostella with an LC50 of 0.978 OB/cm2.